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Sinorhizobium meliloti is a gram-negative soil bacterium found either in free-living form or as a nitrogenfixing endosymbiont of a plant structure called the nodule. Symbiosis between S. meliloti and its plant host alfalfa is dependent on bacterial transcription of nod genes, which encode the enzymes responsible for synthesis of Nod factor. S. meliloti Nod factor is a lipochitooligosaccharide that undergoes a sulfate modification essential for its biological activity. Sulfate also modifies the carbohydrate substituents of the bacterial cell surface, including lipopolysaccharide (LPS) and capsular polysaccharide (K-antigen) (R. A. Cedergren, J. Lee, K. L. Ross, and R. I. Hollingsworth, Biochemistry 34:4467-4477, 1995). We utilized the genomic sequence of S. meliloti to identify an open reading frame, SMc04267 (which we now propose to name lpsS), which encodes an LPS sulfotransferase activity. We expressed LpsS in Escherichia coli and demonstrated that the purified protein functions as an LPS sulfotransferase. Mutants lacking LpsS displayed an 89% reduction in LPS sulfotransferase activity in vitro. However, lpsS mutants retain approximately wild-type levels of sulfated LPS when assayed in vivo, indicating the presence of an additional LPS sulfotransferase activity(ies) in S. meliloti that can compensate for the loss of LpsS. The lpsS mutant did show reduced LPS sulfation, compared to that of the wild type, under conditions that promote nod gene expression, and it elicited a greater number of nodules than did the wild type during symbiosis with alfalfa. These results suggest that sulfation of cell surface polysaccharides and Nod factor may compete for a limiting pool of intracellular sulfate and that LpsS is required for optimal LPS sulfation under these conditions. Symbioses between leguminous plants and the genera Rhizobium, Bradyrhizobium, Mesorhizobium, Azorhizobium, and Sinorhizobium (collectively called rhizobia) result in the formation of a novel plant organ, referred to as the nodule. Within the nodule, differentiated intracellular forms of rhizobia called bacteroids reduce molecular dinitrogen to ammonia. To gain entry into the plant, the bacteria induce morphological alterations of epidermal cells called root hairs, eliciting the formation of a curled structure referred to as a shepherd's crook. Shepherd's crook formation is followed developmentally by the formation of a tubular ingrowth of the root hair, called an infection thread. The infection thread is occupied by rhizobia and penetrates into the root, allowing bacterial entry into the plant. The bacteria within the infection thread are then released into the plant cytoplasm where they develop into nitrogen-fixing bacteroids (3,4,16,20,32,43

We define chromosomal replication complexity (CRC) as the ratio of the copy number of the most replicated regions to that of unreplicated regions on the same chromosome. Although a typical CRC of eukaryotic or bacterial chromosomes is 2, rapidly growing Escherichia coli cells induce an extra round of replication in their chromosomes (CRC = 4). There are also E. coli mutants with stable CRC6. We have investigated the limits and consequences of elevated CRC in E. coli and found three limits: the "natural" CRC limit of 8 (cells divide more slowly); the "functional" CRC limit of 22 (cells divide extremely slowly); and the "tolerance" CRC limit of 64 (cells stop dividing). While the natural limit is likely maintained by the eclipse system spacing replication initiations, the functional limit might reflect the capacity of the chromosome segregation system, rather than dedicated mechanisms, and the tolerance limit may result from titration of limiting replication factors. Whereas recombinational repair is beneficial for cells at the natural and functional CRC limits, we show that it becomes detrimental at the tolerance CRC limit, suggesting recombinational misrepair during the runaway overreplication and giving a rationale for avoidance of the latter. KEYWORDS overinitiation; hydroxyurea; seqA; rep; recA E UKARYOTIC and prokaryotic chromosomes differ in many important aspects (Kuzminov 2014), and one key difference lies in the spatio-temporal organization of chromosomal replication. In contrast to eukaryotes, which perform multibubble replication (Masai et al. 2010), most bacteria replicate their singular chromosome in the unibubble format by initiating bidirectional replication from a designated replication origin (oriC) (Sernova and Gelfand 2008;Leonard and Méchali 2013) and finishing replication within a broad termination zone (ter) (Mirkin and Mirkin 2007;Duggin et al. 2008). Eukaryotes always perform a single replication round in their chromosomes (Masai et al. 2010;Diffley 2011), keeping the ratio of maximally replicated to unreplicated DNA in the same chromosome (the "replication complexity index") strictly at 2 ( Figure 1A). Due to the defined origin and terminus of prokaryotic chromosomes, chromosomal replication complexity in bacteria can be simply expressed as the ori/ter ratio ( Figure 1B). Even though unique cell cycles in some bacteria, such as Caulobacter, also maintain a strict CRC = 2 (Collier 2012), bacterial cells are generally recognized for their ability to support multiple concurrent replication rounds within the same chromosome (Morigen et al. 2009).In reality, under typical growth conditions the ori/ter ratio in exponentially growing bacterial cells is still 2 (Bird et al. 1972;Bipatnath et al. 1998;Wang et al. 2007;Murray and Errington 2008;Rotman et al. 2009;Stokke et al. 2011), showing that bacterial cells, like eukaryotes, also prefer to deal with a single replication round in their chromosomes. However, due to the peculiarity of the prokaryotic chromosome cycle (Kuzminov 2013), whe...

In vitro, purified replisomes drive model replication forks to synthesize continuous leading strands, even without ligase, supporting the semidiscontinuous model of DNA replication. However, nascent replication intermediates isolated from ligase-deficient Escherichia coli comprise only short (on average 1.2-kb) Okazaki fragments. It was long suspected that cells replicate their chromosomal DNA by the semidiscontinuous mode observed in vitro but that, in vivo, the nascent leading strand was artifactually fragmented postsynthesis by excision repair. Here, using high-resolution separation of pulse-labeled replication intermediates coupled with strand-specific hybridization, we show that excision-proficient E. coli generates leading-strand intermediates >10-fold longer than lagging-strand Okazaki fragments. Inactivation of DNA-repair activities, including ribonucleotide excision, further increased nascent leading-strand size to ∼80 kb, while lagging-strand Okazaki fragments remained unaffected. We conclude that in vivo, repriming occurs ∼70× less frequently on the leading versus lagging strands, and that DNA replication in E. coli is effectively semidiscontinuous.